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Plant Molecular Biology 15: 513-516, 1990.
© 1990 Kluwer Academic Publishers. Printed in Belgium.
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Plant Molecular Biology Update
RFLP mapping of the abpl locus in maize (Zea mays L.)
Marian L6bler and Ann M. Hirsch
University of California Los Angeles, Department of Biology, 405 Hilgard Ave, Los Angeles, CA 90024,
USA
Received and accepted 12 June 1990
The plant hormone auxin controls a wide variety
of growth and differentiation processes in higher
plants. The molecular mechanism by which this
control is exerted is essentially unknown. Auxin
binds specifically to an auxin-binding protein
(ABP) which is thought to be the first element in
a signal transduction pathway [7]. However, the
other components of an auxin-activated signal
transduction pathway, which lead to the biological
response, have not been identified. If mutations in
the presumed signal transduction pathway could
be obtained, they would be extremely useful in
elucidating the molecular mechanism of auxin
action.
Auxin biosynthesis and auxin response mutants
have been described in some dicotyledonous
plants [6, 9]. That such mutants are found at
frequencies 10-100 times lower than null mutants
of other loci [3, 6], e.g. starch or lipid biosynthesis
[6], strongly suggests that null mutants related to
auxin physiology are lethal [8]. The mutants that
have been characterized show pleiotropic effects
and hence a variety ofphenotypes. By determining
the genetic locus of at least one component of the
auxin signal transduction pathway, we should be
able to characterize auxin response phenotypes
better. This will allow us to look for similar
phenotypes with the goal of finding mutants in the
signal transduction pathway. We have used
RFLP mapping (restriction fragment length polymorphism mapping) to assign a locus to the ABP,
the putative first element in the auxin signal transduction pathway.
Maize recombinant inbred lines from the F2
populations of TXCM as well as the parental lines
T232 and CM37 [ 1] were used to prepare genomic
D N A from leaves. The isolation of an ABP
c D N A clone (pBS.ABP) is described elsewhere
[ 10]. All restriction enzymes and the nick translation kit were purchased from Bethesda Research
Laboratory, Bethesda, MD. D N A was extracted
from young leaves (approx. 1 g of tissue) following
a procedure of Cone [2] with some modifications.
In brief, leaf tissue frozen on dry ice at the time
of harvest was homogenized in liquid nitrogen
with pestle and mortar. The resulting fine powder
was added to 5 ml homogenization buffer (7 M
urea, 0.35 M NaC1, 0.05 M EDTA, 0.01 M TrisHC1 pH 7.6, 2% sodium lauroylsarcosine) in a
glass scintillation vial and rocked on a shaker
until it appeared homogeneous (ca 15 min). An
equal volume of phenol, equilibrated with homogenization buffer, was added and again shaken
until apparent homogeneity (ca 15 min). The
highly viscous slurry was centrifuged and the
aqueous phase was extracted with an equal
volume of chloroform : isoamyl alcohol (24 : 1).
D N A was precipitated by adding sodium acetate
(final concentration 0.2 M) and 0.55 volumes of
isopropanol. High molecular weight D N A was
spooled out with a bent pasteur pipette and resuspended in 4 ml TE (10 m M Tris-HCl pH 8, 1 mM
EDTA). A final ethanol precipitation in the
presence of 2 M ammonium acetate yielded a
nucleic acid preparation that could be digested
with restriction enzymes.
Restriction digests were performed according
to manufacturer's protocols using 1.5 ~g of D N A
and 20 units of enzyme for a 3 h incubation time
at the appropriate temperature. Restriction fragments were separated on 0.8~ agarose gels and
blotted onto nitrocellulose (Schleicher & Schuell)
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Fig. 1. Southern blot of maize genomic D N A from the parental lines T232 and CM37 indicated by T and CM respectively. The
restriction enzymes used are indicated above the lanes. Nick translated ABP c D N A was used as a hybridization probe.
Fig. 2. Southern blot of maize genomic Sst I D N A fragments from the recombinant inbred F2 families (TXCM) and the parental
lines T232 and CM37. Nick translated ABP c D N A was used as a hybridization probe. Note that the observed hybridization
pattern of the recombinant inbreds matches either one of the parental lines (T232 or CM37).
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or GeneScreen (BioRad) membranes according
to manufacturers' protocols. Blots were hybridized to a nick-translated probe of pB S.ABP insert
overnight at 37 °C in the presence of 50~o
formamide. After the blots had been washed with
0.2 × SSC (30 mM NaCI, 3 mM sodium citrate)
containing sodium lauroylsarcosine (0.05 ~o) and
pyrophosphate (0.01 ~o) at 55 °C, they were exposed to X-ray film for 16 h. Nick translation was
performed according to the protocol provided by
BRL. For the Southern hybridizations we followed the protocols recommended by the manufacturers of the transfer membranes. B. Burr
analyzed the RFLP data by comparison to the
RFLP database for maize [ 1].
In the homozygous parental lines T232 and
CM37, one or two genomic restriction fragments
hybridize to the ABP cDNA (Fig. 1), suggesting
that there is only a single gene for the auxinbinding protein in maize. On a Southern blot
developed under low stringency conditions, no
additional bands specific for the ABP can be
detected (data not shown). Of the restriction
enzymes used to digest the genomic DNA, only
Eco RI cuts the ABP cDNA, yielding two fragments similar in size [10]. When genomic DNA
is probed, we detect only one Eco RI restriction
fragment which hybridizes to the ABP cDNA
(Fig. 1). The signal intensity of this particular
band is only half of the signal intensity of other
restriction fragments (Hind III and Kpn I). We
therefore conclude that one half of the ABP gene
is digested by Eco RI to very short fragments
which are not contained on the blot. Because only
one Eco RI fragment and two restriction fragments of the Barn HI, Bgl II, Sca I, Sph I and
Sst I digests of the genomic DNA hybridize to the
ABP cDNA (Fig. 1) we conclude that introns are
present in the abpl gene.
A restriction fragment length polymorphism
(RFLP) was found between the parental lines
T232 and CM37 in the restriction digests
Hind III, Sca I, Sph I, and Sst I (Fig. 1). Because
the Sst I RFLP could be scored most easily, Sst I
was chosen to digest DNA from the recombinant
inbreds derived from the F2 generation.
Southern hybridization with the ABP cDNA
probe revealed the RFLP pattern shown in Fig. 2.
A locus was assigned to the ABP cDNA clone by
computer analysis of the data presented in Fig. 2.
It maps to the long arm of chromosome 3 at
position 70. We name this locus abpl for several
reasons. First, the analysis of the cDNA clones
isolated by different groups [4, 5, 10] reveals that
the ABP is a soluble protein and lacks transmembrane domains as one would expect for a
receptor protein. Second, the publications concerning the ABP cDNA have used ABP to name
the isolated clones [5, 10], with the exception of
Hesse et al. [4] who used axr-1. Third, we wish
to avoid confusion with auxin resistance genes
(,4xr-1) described by Estelle and Sommerville [3].
The abpl locus lies within a chromosomal region
that is rich in genetically characterized develop-
m
20
e8
8,15
.
8.35
61
-
71
-- ¢% p d g 2 ~
--
tpi4
6.06
abpl
5,37
8.01
10.24A
-""
138
141
15.20
6.16
3.18
1.297
mdh3
---
al
--
npi425
Fig. 3. Maize c h r o m o s o m e 3 with R F L P marker locations
and the abpl locus (triangle). The c h r o m o s o m e m a p was
r e d r a w n from reference 1.
516
mental mutants [ 11]. However, we do not know
if the abpl locus is identical to any of the known
genetic marker loci.
4.
Acknowledgements
We would like to express our gratitude to R.J.
Schmidt from UC San Diego who let us pick leaf
samples from the maize genotypes used in this
study, to B. Burr from Brookhaven Natl. Laboratory who compared the RFLP data with the database, and to B.O. Phinney and S. Kirchanski for
critically reading the manuscript. This work was
supported by a N S F grant to A.M.H. (DCB
8703297).
5.
6.
7.
8.
References
1. Burr B, Burr FA, Thompson KH, Albertsen MC, Stuber
CW: Gene mapping with recombinant inbreds in maize.
Genetics 118:519-526 (1988).
2. Cone K: Yet another rapid plant DNA prep. Maize
Genetics Cooperation News Letter 63:68 (1989).
3. Estelle MA, Sommerville C: Auxin-resistant mutants of
9.
10.
11.
Arabidopsis thaliana with an altered morphology. Mol
Gen Genet 206:200-206 (1987).
Hesse T, Feldwisch J, Balsh~isemann D, Bauw G, Puype
M, Vandekerckhove J, L6bler M, Kl~tmbt D, Schell J,
Palme K: Molecular cloning and structural analysis of a
gene from Zea mays (L.) coding for a putative receptor
for the plant hormone auxin. EMBO J 8:2453-2461
(1989).
Inohara N, Shimomura S, Fukui T, Futai M: Auxinbinding protein located in the endoplasmic reticulum of
maize shoots: Molecular cloning and complete primary
structure. Proc Natl Acad Sci USA 86:3564-3568
(1989).
Last RL, Fink GR: Tryptophan-requiringmutants of the
plant Arabidopsis thaliana. Science 240:305-310 (1988).
L6bler M, Kl~imbt D: Auxin-binding protein from
coleoptile membranes of corn (Zea mays L.) II. Localization of a putative auxin receptor. J Biol Chem 260:
9854-9859 (1985).
Mirza JI, Maher EP: More 2,4-D resistant mutants.
Arabidopsis Inf Serv 17:103-107 (1980).
Scott IM: Plant hormone response mutants. Physiol
Plant 78:147-152 (1990).
Tillmann U, Viola G, Kayser B, Siemeister G, Hesse T,
Palme K, L6bler M, Kl~mbt D: cDNA clones of the
auxin-binding protein from corn coleoptiles (Zea
mays L.): isolation and characterization by immunological methods. EMBO J 8:2463-2467 (1989).
Maize Genetics Cooperation Newsletter 64 (1990).